The long term goals of our research are to characterize the CNS myelin radial component, which is a multilamellar super-assembly of claudin 11 tight junctions (TJs) in compact regions of myelin sheaths. To do this, we will identify oligodendrocyte proteins that associate with claudin 11, which is the major membrane protein and is necessary for radial component assembly. We have defined the electrophysiology of the radial component in vivo, by mathematical modeling, and we have shown its physiological properties by X-ray and neutron diffraction. Most recently, we demonstrated that absence of the radial component leads to behavioral phenotypes in amygdala and auditory brainstem circuits. Currently, claudin 11 is the only canonical tight junction protein known to localize to the radial component. Moreover, no other canonical tight junction proteins are present in the radial component, indicating that this structure is almost completely novel in terms of its protein composition because loss of claudin 11 abolishes formation of the radial component. To identify and characterize radial component associated proteins, we will use BioID technology, which has been used in several in vitro systems to identify unknown proteins in macromolecular complexes. We have taken this technology a step further by generating transgenic mice that harness this technology in vivo. This step is necessary because current cell culture systems are not sufficiently robust to enable CNS myelinogenesis in vitro. In the preliminary data, we show that we have generated a mouse with BioID technology incorporated into the Claudin 11 gene. We show that this technology is expressed by oligodendrocytes and present in purified myelin membranes from spinal cord. Finally, we show that BioID is functional under appropriate conditions and we observe a number of unknown BioID-biotinylated proteins (candidate radial component proteins) ranging from 20-200 kDa. In Aim#1, we will purify these radial component proteins for mass spectrometry using 2 complimentary experimental approaches, BioID labeling and immunoprecipitation with claudin 11 antibodies. In addition, we will test that the candidate proteins identified actually colocalize with claudin 11 in tissue sections from mouse brain and primary oligodendrocyte cultures. We will also immunoprecipitate these proteins to show they pulldown claudin 11. In Aim#2, we will clone cDNAs encoding the candidate proteins and express them in oligodendrocyte cell lines to determine if they colocalize with claudin 11. We will also immunoprecipitate these proteins to show they pulldown claudin 11.